The physiological effects of the hormone insulin are mediated by the insulin receptor, a member of a large family of cell surface receptors with intrinsic tyrosine kinase activity. This family also includes the receptors for epidermal growth factor (EGF), fibroblast growth factor (FGF), and nerve growth factor (NGF), among others. Activation of receptor tyrosine kinases upon ligand binding is achieved through autophosphorylation of specific tyrosine residues in the cytoplasmic domain. For the insulin receptor and many other receptor tyrosine kinases, autophosphorylation both stimulates tyrosine kinase activity and provides binding sites for downstream signaling proteins that recognize phosphotyrosine in particular sequence contexts. The specific aims of this proposal are: 1. Determination of the molecular basis of insulin receptor activation via tyrosine autophosphorylation. 2. Elucidation of the structural determinants of insulin receptor substrate specificity. 3. Molecular characterization of the interactions between the autophosphorylated insulin receptor and downstream signaling proteins. X-ray crystallographic studies of the insulin receptor tyrosine kinase in different phosphorylation states and in complex with substrate peptides and downstream signaling proteins will be the primary means to accomplish these aims. The information gained from this study will enhance our knowledge of insulin receptor signaling and provide a structural framework for understanding the deleterious effects of a number of insulin receptor mutations that lead to non-insulin-dependent diabetes mellitus. The results will also be applicable to many growth factor receptors which mediate cell growth and differentiation. High-resolution structures of the insulin receptor tyrosine kinase should prove useful in the design of small molecule inhibitors targeted against tyrosine kinases that contribute to cancer.